Development of a smart pH-responsive nano-polymer drug, 2-methoxy-4-vinylphenol conjugate against the intestinal pathogen, Vibrio cholerae

Vibrio cholerae causes cholera, an acute diarrhoeal disease. The virulence in V. cholerae is regulated by the quorum-sensing mechanism and response regulator LuxO positively regulates the expression of virulence determinants adhesion, biofilm formation, and cholera toxin production. Previous in-silico studies revealed that 2-methoxy-4-vinylphenol could bind to the ATP binding site of LuxO and the complex was compact and stable in pHs like intestinal pHs. Here, we have explored the polymeric nano-formulation of 2-methoxy-4-vinylphenol using cellulose acetate phthalate for controlled drug release and their effectiveness in attenuating the expression of V. cholerae virulence. Physico-chemical characterization of the formulation showed particles with a mean size of 91.8 ± 14 nm diameter and surface charge of − 14.7 ± 0.07 mV. The uniform round polymeric nanoparticles formed displayed about 51% burst release of the drug at pH 7 by 3rd h, followed by a controlled linear release in alkaline pH. The polymeric nanoparticles demonstrated a tenfold increase in intestinal membrane permeability ex-vivo. At lower concentrations, the 2-methoxy-4-vinylphenol polymeric nanoparticles were non-cytotoxic to Int 407 cells. In-vitro analysis at pH 6, pH 7, pH 8, and pH 9 revealed that cellulose acetate phthalate—2-methoxy-4-vinylphenol nanoparticles were non-bactericidal at concentrations up to 500 μg/mL. At 31.25 μg/mL, the nanoparticles inhibited about 50% of the biofilm formation of V. cholerae MTCC 3905 and HYR14 strains. At this concentration, the adherence of V. cholerae MTCC 3905 and HYR14 to Int 407 cell lines were also significantly affected. Gene expression analysis revealed that the expression of tcp, qrr, and ct at pH 6, 7, 8, and 9 has reduced. The CAP-2M4VP nanoparticles have demonstrated the potential to effectively reduce the virulence of V. cholerae in-vitro.

(2M4VP) with cellulose acetate phthalate (CAP) was prepared by a modified nanoprecipitation method. About 10 mg of CAP was dissolved in 1 mL of acetone and 10 mg of 2M4VP in 1 mL of methanol. Both the drug and the polymer preparation were mixed to obtain the organic phase. About 0.5% of Pluronic® F127 in distilled water was prepared to form the aqueous phase. The 2 mL of the organic phase was added dropwise into the aqueous media using a syringe and the dispersion was left under constant and continuous stirring at 700 RPM at room temperature until the organic phase completely evaporates. The colloidal aqueous solution containing the nanoprecipitated particles of CAP-2M4VP was then freeze-dried by lyophilization 14 (Supplementary Fig. 1). Material characterization. The particle size distribution and stability of the CAP-2M4VP formulation were measured by the dynamic light scattering (DLS) and zeta potential using Zetasizer (NanoZs, Malvern Instruments, UK). The chemical interaction of drug and polymer present in the CAP-2M4VP particles was analyzed by Fourier-transform infrared spectroscopy (FTIR). The freeze-dried samples were placed on a hole of 1 mm on a stell gasket of 0.05 mm thickness on top of a diamond anvil cell. Then the sample was transferred to a stub and scanned from wavelength 4000 cm −1 to 400 cm −115 . The diffraction pattern of the CAP-2M4VP and 2M4VP were compared by X-ray diffractometer with Cu-Kα radiation at 30 mÅ of current and 40 kV volatage (Rigaku, Japan). The analysis was performed by scanning the samples at 2θ from 0° to 100°1 6 . The variation in the thermal properties of the polymeric nanoparticles compared to the pure drug was studied using Thermogravimetry and Differential Scanning Calorimetry (NETZCH, Germany). About 5-10 mg of the samples were individually placed in an aluminum pan and heated at the rate of 10 °C/min up to 1000 °C and the result was represented as weight loss and heat flow against temperature. The surface morphology of the lyophilized CAP-2M4VP nanoparticles preparation and a pure sample of 2M4VP was captured after gold sputter-coating using Scanning Electron Microscope (VEGA3, TESCAN Analytics, Czech Republic). www.nature.com/scientificreports/ Entrapment efficiency. The percentage of 2M4VP entrapped within the nanoparticles was studied by centrifuging the CAP-2M4VP nanosuspension preparation at 12,000 rpm at 4°C for 20 min. The total amount of 2M4VP entrapped within the CAP nanoparticles was determined by the estimation of unentrapped free drug present in the supernatant. The absorbance of the supernatant solution was measured at 268 nm using respective blank. The percentage of entrapment efficiency was calculated by the following formula 17 , Drug content. The total amount of 2M4VP present within the CAP nanoparticles suspension was analyzed.
About 1 mL of the nanosuspension was dissolved in 9 mL of methanol and left for 24 h at 4 °C. The solution was sonicated and the amount of drug present was measured using a UV-Visible spectrophotometer at 268 nm using a respective blank 18,19 .
In-vitro drug release studies. The percentage of drug release of 2M4VP encapsulated in CAP nanosuspension was studied in the presence of dissolution media-buffers at various pHs (1.2, 5.8, 7, 8 and 9). About 1 mg/mL of the CAP-2M4VP nanoparticles were loaded into a dialysis membrane (HiMedia, Mumbai, India; Pore size: up to 14,000 Daltons) and the ends were sealed. The membrane was then placed inside a screw cap bottle filled with 5 mL of dissolution media of specific pH. The set-up was placed under constant stirring at 200 RPM at 37 °C. At regular time intervals, 5 mL of samples were removed using a syringe and were replaced with the 5 mL of the respective fresh buffers. The samples collected were analyzed at 268 nm using a UV-Visible spectrophotometer using respective blank. The study was performed in triplicate and presented as the mean with standard deviation limits 19 .
Drug release kinetics. The cumulative percentage of drug release profiles obtained for pH 1.2, 5.8, 7.0, 8.0, and 9.0 was further analyzed by fitting the data into drug release kinetics models. The data from in-vitro drug release was fitted into zero-order, first-order, Higuchi, Korsmeyer-Peppas, Hixson-Crowell, Hopfenberg, Baker-Lonsdale, Weibull, and Gompertz kinetic models. The values of R 2 , Rate Constant (K), Sum of Squared Residual (SSR) and Release exponent (n) were identified for CAP-2M4VP nanoparticles at each pH included in this study 19 .
Ex-vivo permeation assay. The permeation of CAP-2M4VP nanoparticles across excised goat intestinal mucosal membrane ex-vivo was performed using the Franz cell diffusion chamber. The Franz cell diffusion chamber comprises two compartments-an upper donor connected to a lower acceptor (receptor) compartment. The capacity of the acceptor compartment is approximately 15 mL and the available diffusion area is 1.8 cm 2 in diameter. The acceptor compartment was filled with phosphate buffer pH 7 and a magnetic bead was placed inside. The 5 × 5 cm goat intestinal mucosal tissue was mounted on top of the acceptor compartment with the mucosal region facing towards the donor compartment with constant contact with the phosphate buffer pH 7 so that bubbles formation was prevented. The donor compartment was then placed on top of the intestinal tissue and both the compartment were held tightly in position with rubber clamps. The donor compartment was filled with phosphate buffer pH 7 containing 1 mg/mL of the CAP-2M4VP nanosuspension preparation. The opening of the donor compartment was covered with aluminium foil to prevent evaporation. The entire setup was placed on a magnetic stirrer at 50 RPM and continuously stirred at room temperature. About 500 μL of samples were collected from the receptor compartment at every 30 min interval for the first 2 h and then at 1 h interval for 8 h continuously. The cumulative amount of drug permeation was determined by a UV-Visible spectrophotometer at 268 nm. The volume withdrawn from the receptor compartment was replaced by the aliquoting same volume of the phosphate buffer into the donor compartment. The permeation data analysis was performed as per described by Ramyadevi and Rajan 19 . The results presented a mean ± SD of experiments performed in triplicates.
Cell viability assay. The effect of the CAP-2M4VP nanoparticles on host cell viability was assayed using Int 407 cell lines, cultured in minimal essential medium (MEM) supplemented with 10% fetal bovine serum (FBS), 1% penicillin/streptomycin (w/v), and 1% l-glutamine using the MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay. The Int 407 cell lines were procured from National Centre for Cell Science (NCCS), Pune, India. About 1 × 10 4 Int 407 cells/well were seeded in a 96-well microtiter plate and incubated at 37 °C in a CO 2 incubator for 24 h. After the incubation period, the cells were treated with various concentrations (100 μg/mL, 50 μg/mL, 25 μg/mL, 12.5 μg/mL, 6.25 μg/mL) of CAP-2M4VP and incubated for another 24 h at 37 °C in a CO 2 incubator. After 24 h, 1 mg/mL of MTT was added, and the microtiter plate was incubated at 37 °C for 3 h. Dimethyl sulfoxide (DMSO) was added to dissolve the formazan crystals, and absorbance was read at 570 nm using a microplate reader spectrophotometer (Synergy H1) 20 . www.nature.com/scientificreports/ absorbance at 595 nm using a microtitre plate reader (iMark, BIORAD, Japan). Experiments were performed in triplicates and the data are reported as mean with standard deviation 21 .

Inhibition of growth.
Inhibition of biofilm formation. Fluorescent microscopy of biofilm. The inhibition of biofilm formation by CAP-2M4VP against V. cholerae MTCC 3905 and HYR14 was observed using fluorescent microscopy. About 25 mL of LB media adjusted to pH 6, pH 7, pH 8, and pH 9 containing 31.25 μg/mL of CAP-2M4VP were aliquoted to 50 mL sterile screw capped tubes. One percent of the diluted (1:100 (OD 595 = 0.2)) overnight culture of V. cholerae strains MTCC 3905 and HYR14 were added to the tubes. Sterile glass slides were placed inside the tubes and incubated at static conditions for 24 h at 37 °C. pH-adjusted media tubes without CAP-2M4VP served as control. After the incubation period, the slides were recovered from the tubes and rinsed with sterile 0.9% NaCl to remove planktonic cells and media. As per the manufacturer's protocol, the adherent biofilm was stained with BacLight Bacterial Viability Kit (ThermoFisher Scientific, USA). The images of biofilm were observed under a 40× objective using a fluorescent microscope (Nikon Eclipse Ts2) 23 .
Cell adhesion assay. Int 407 cell lines were grown up to 90% confluency using MEM supplemented with 10% FBS without antibiotics at 37 °C in a 5% CO 2 incubator. The cells were washed with phosphate-buffered saline at pH 7 and fresh media was added. The overnight culture of V. cholerae MTCC 3905 and HYR14 strains at a concentration of multiplicity of infection of 50 (MOI 50) was added along with 31.25 μg/mL of CAP-2M4VP. The plates were incubated at 37 °C in 5% CO 2 for 1 h. After the incubation period, the cells were washed with PBS to remove the non-adherent bacteria. The cells were lysed using 0.1% of Triton X-100 prepared in PBS. The CFU of the adherent bacteria was enumerated and presented as a percentage of bacteria adherent to Int 407 cell lines. The experiments were performed in triplicates 24 . For imaging using light microscopy, the Int 407 cells were grown in Petri plates with sterile coverslips in MEM medium containing 10% FBS without antibiotics at 37 °C in a 5% CO 2 atmosphere. The infection procedure was followed and after the infection period, the coverslips were removed and fixed with methanol for 30 min followed by staining with Giemsa stain for 20 min. The stained cells were washed with PBS and air-dried. The bacteria adherent to Int 407 cells were observed at 40× magnification using a microscope 25 .

Gene expression analysis by quantitative RT-PCR.
Gene expression analysis to understand the effect of CAP-2M4VP on the V. cholerae MTCC 3905 cells was performed using quantitative real-time PCR. RNA was extracted from 24 h culture of V. cholerae MTCC 3905 cells treated with 31.25 μg/mL of CAP-2M4VP following the manufacturer's guidelines of the HiMedia RNA Extraction kit (MB613) (HiMedia, India). The purity and integrity of the isolated RNA were analyzed using NanoDrop (Thermo Fisher Scientific, United States). The conversion of total RNA to cDNA was performed using the iScript™ cDNA Synthesis kit following the manufacturer's guidelines. The effect of CAP-2M4VP on the gene expression in MTCC 3905 was investigated using qRT-PCR using Real-Time PCR System (Applied Biosystems, USA). The primers used are described in Supplementary Table 1. 16S rRNA served as reference gene and the expression of genes was calculated using the 2 -ΔΔ CT method. The PCR conditions set were as described previously 26 .
Statistical analysis. GraphPad Prism software version 8.0.2 (GraphPad Software Inc., San Diego, CA, United States) was used for statistical analysis of the results. The results were expressed as mean ± standard deviations (SD) and all the experiments were performed in triplicates. The significance was analyzed by Oneway ANOVA followed by Dunnett's multiple comparison test and Student t-test with p set at p ≤ 0.05.

Results
Nanoparticles characterization. The physicochemical characterization of CAP-2M4VP nanoparticles at neutral pH revealed that the particles had a mean size of 91.8 ± 14 nm in diameter. The polydispersity index (PDI) was 0.251 ± 0.07, and the zeta potential is − 14.7 ± 0.07 mV indicating that the nanoparticles have good size distribution and are negatively charged ( Supplementary Fig. 2a,b). Scanning electron microscopy images revealed that the pure drug (2M4VP) has an irregular flakes-like structure and the size ranged from 0.9 to 2.6 μM in size (Fig. 1a). The optimized formulation of CAP-2M4VP nanoparticles was smooth with uniform www.nature.com/scientificreports/ spherical structures. The size of the nanoparticles ranged between ~ 300 and ~ 400 nm (Fig. 1b). The total drug content in the optimized CAP-2M4VP nanosuspension was about 79.52 ± 0.2% and the highest entrapment efficiency of the nanoparticles was 59.7 ± 2%.
Interaction between the polymer CAP and the drug 2M4VP. The FTIR spectrum of pure compound 2-methoxy-4-vinylphenol had displayed the characteristic peaks corresponding to its functional groups viz., at 3416 cm −1 for -OH stretching of alcohol, 2927 cm −1 for -C-H stretching and 1606 cm −1 for -C=C stretching of alkene double bond, 1450 cm −1 at -C-H bending of a methyl group, 1269 cm −1 and 1230 cm −1 for -C-O stretching and 1364 cm −1 for -OH bending. Therefore, the purity and chemical nature of the compound were confirmed (NIST Chemistry WebBook, SRD 69, WILEY SpectraBase™). In the case of the polymer Cellulose Acetate Phthalate, the peaks were obtained at 3439 cm −1 corresponding to -OH stretching of the alcoholic group, 2925 cm −1 for -C-H stretching of aliphatic chain, 1728 cm −1 for -C=O stretching, 1249 cm −1 for -C-O stretching, 1384 cm −1 for -OH bending and 742 cm −1 for -C=C bending frequency. The specific peaks at the defined wavenumbers represented the characteristic identity and chemical nature of the polymer material. The formulated nanoparticles of 2M4VP using CAP polymer had shown peaks at 3439 cm −1 representing -OH stretching of the alcoholic group, 2917 cm −1 for -C-H stretching, 1741 cm −1 for -C=O stretching, 1637 cm −1 for -C=C stretching, 1250 cm −1 for -C=O stretching, 1466 cm −1 for -C-H bending, and 1374 cm −1 for -OH bending (Fig. 2). The characteristic peaks of both drug and polymer molecules were displayed by the nanoparticles, wherein the functional groups and features of the drug were predominant without significant alterations in the chemical nature and stability/purity of the active compound.
Crystal behaviour changes of 2M4VP within CAP. The XRD pattern of the pure 2M4VP displayed sharp peaks with two high-intensity points between the range of 20-30 in the 2-theta scale, representing the characteristic crystalline nature of the drug. In the case of the XRD spectrum of 2M4VP loaded CAP nanoparticles, the crystalline peaks of the drug were not visualized due to encapsulation of 2M4VP in the CAP polymer matrix. This solid-state conversion of the drug in addition to amorphous CAP polymer confirmed the stability and entrapment of drug in the nanoparticles 27 (Fig. 3). The transition of crystalline to amorphous nature in nanoparticle formulation could support uniform distribution, enhanced dissolution, and uptake of the drug in-vivo.
Increased thermal stability of 2M4VP encapsulated within the polymer CAP. The DSC thermogram of a pure sample of 2M4VP showed an endothermic peak at the temperature range of 194.43 °C, measured as the melting point of the crude sample, which could be confirmed by the corresponding sudden weight loss from 100 to 40% in the TGA curve. Further increase in temperature led to decomposition of the sample with a residual weight of 32% (Fig. 4a). In the case of CAP-2M4VP nanoparticles, the endothermic peak of the drug sample was diminished by the presence of amorphous CAP polymer, which exhibited thermal stability up to  (Fig. 4b). The results confirmed the formation of stable amorphous nanoparticles of 2M4VP uniformly dispersed within CAP polymer.
In-vitro controlled release of 2M4VP from CAP.   Mechanism of 2M4VP release from CAP. The cumulative drug release percentage data obtained from in-vitro drug release studies were analyzed for the rate of release and mechanism using kinetic models including zero order, first order, Higuchi, Korsmeyer-Peppas, Hixson-Crowell, Hopfenberg, Baker-Lonsdale, Weibull and Gompertz. The mechanism of diffusion of the drug from the nanoparticles was confirmed by Korsemeyer-Peppas and Weibull models, where the plots were linear with R 2 values ranging between 0.97 and 0.99 at all pHs. The best fit model was the Korsmeyer-Peppas model, as the R 2 values were greater than 0.97 at all pHs. This indicated that the mechanism of drug release was diffusion controlled. The n-value less than 0.20 explained the Fickian transport kinetics for the diffusion of the drug from nanoparticles. The Weibull kinetics model confirmed the drug release pattern based on the matrix type of systems, herein the 2M4VP loaded CAP matrix nanoparticles (Supplementary Table 2).

Ex-vivo permeation of CAP-2M4VP. The formulated nanoparticles CAP-2M4VP showed about
119.0713 µg/cm 2 permeation through the intestinal mucosal membrane within 2 h. Further, there was a linear increase from 3 h to 8 h to reach the cumulative permeation amount of 184.8705 ± 47.93 µg/cm 2 . The nano-formulation showed a steady-state flux of 11.15 ± 4.64 µg/cm 2 /h and permeability coefficient of 0.0111 ± 0.004 cm/h × 10 -3 across the goat intestinal membrane (Fig. 6). Meanwhile, for the aqueous dispersion of pure 2M4VP, only 18.48 ± 6.67 µg/cm 2 cumulative amount of drug was permeated across the intestinal mucosal membrane at the end of the 8th h. When compared to the pure 2M4VP permeation across intestinal membrane, the CAP-2M4VP

CAP-2M4VP is non-cytotoxic at lower concentrations. The effect of the CAP-2M4VP formulation
on the viability of Int 407 cells was investigated by MTT assay. The data shows that at lower concentrations ranging from 50 to 6.25 μg/mL the polymeric nano-formulation did not affect the viability of the Int 407 cell lines. At 100 μg/mL, the Int 407 cell viability was significantly affected (Fig. 7). This indicated that the polymeric CAP nano-formulation of 2M4VP is non-cytotoxic at a lower concentration.    (Fig. 11a,b) and HYR14 strains (Fig. 11d,e) showed a reduction in bacterial cell adherence. Enumeration of V. cholerae MTCC 3905 and HYR14 adhered to Int 407 cell lines revealed that upon treated with CAP-2M4VP (31.25 μg/mL), the percentage of V. cholerae cells attached to Int 407 cell lines were significantly less compared to the untreated Int 407 cells (Fig. 11c,f). Adherence is a key factor in V. cholerae pathogenesis as this leads to effective colonization of the small intestine which in turn allows cholera toxin to reach the epithelium causing cholera 28 . Hence, the display of reduction of adherence of V. cholerae strains to Int 407 cell lines indicates the potential of CAP-2M4VP to affect the initial stage of infection.

Downregulation of virulence by CAP-2M4VP. Gene regulation analysis by qRT-PCR revealed that
CAP-2M4VP at all pHs downregulates the expression of various virulence encoding genes in MTCC 3905. At pH 6, tcp was downregulated by 1 log 10 fold indicating the reduction in the expression of toxin co-regulated pili. At pH 7, 1 log 10 reduction in the expression of aphA, qrr 2, qrr-4, tcp, and ct was noted. At pH 8, 1 log 10 reduction in qrr-4 and tcp and at pH 9, 1 log 10 reduction in qrr-2 was observed. Overall, at pH 6, pH 7, pH 8, and pH 9, downregulation in the expression of genes encoding the response regulator aphA, sRNA qrr 2, and qrr-4, virulence factors including toxin co-regulated pili (TCP) and cholera toxin (CT) were observed (Fig. 12).

Discussion
In our previous study, in-silico analysis revealed that 2-methoxy-4-vinylphenol present in the organic extract of Micromonospora sp. RMA46 could interact with the active site of LuxO of V. cholerae potentially inhibiting the activity of LuxO 11 . V. cholerae has developed a sophisticated acid tolerance response enabling it to survive the gastric acids in the human stomach 29 . Upon escaping the acid barrier, the bacterial cells reach the human www.nature.com/scientificreports/ small intestine, which is the primary site of infection 30 . To facilitate the controlled release of 2M4VP in the small intestinal condition, a pH-dependent polymer cellulose acetate phthalate was used for the preparation of polymeric nanoparticles of 2M4VP. This preparation was analyzed in-vitro for effective controlled drug release and, its potential to attenuate virulence in V. cholerae. The smaller size of the CAP-2M4VP nanoparticles indicated the compatibility of the emulsion evaporation method in the formulation of nanoparticles. The formation of nanoparticles less than 100 nm has been beneficial in terms of gastrointestinal drug delivery. Smaller-sized drug nanoparticles allows increased drug accumulation at the site of infection, improved transport, and retention in the gastrointestinal tract, uniform drug distribution, and increased drug uptake in cells and tissues in the gastrointestinal tract 31 . The zeta potential of the CAP-2M4VP was net negative charge and the polydispersity index was 0.251 ± 0.07 indicating a narrow size distribution of the CAP-2M4VP nanoparticle 32,33 .  www.nature.com/scientificreports/ Good stability of the nanoparticles prevents the release of the drug during storage, reducing the administrative concentration of the drug to induce effective therapeutic activity 34 . Also, nanoparticles with a negative charge have been suggested to have a greater cycle time in-vivo than positively charged nanoparticles 35 . As the mucus is negatively charged, the negative charge of CAP-2M4VP nanoparticles could be beneficial for mucus penetration due to the absence of ionic interaction between the CAP-2M4VP and mucus 36 .
SEM analysis has revealed a transformation of the irregular flakes of the pure drug 2M4VP to more regular spherical-shaped CAP-2M4VP nanoparticles indicating uniformity in the formulation. The FTIR analysis has shown that no functional group has been added or removed from the CAP-2M4VP signifying no loss of functionality of the 2M4VP. XRD displayed the solid-state transition of the 2M4VP from crystalline to an amorphous  www.nature.com/scientificreports/ pattern in CAP-2M4VP. The amorphous XRD pattern is characteristic of cellulose acetate phthalate 27 . The loss of crystalline peaks in XRD analysis by CAP-2M4VP shows that the drug 2M4VP has effectively encapsulated within CAP. Other advantages of amorphous nanoparticle formulation is an increase in the dissolution and bioavailability 16,27,37 . The complete release of the 2M4VP from CAP nano-formulation was observed at pH 7.0 at 40th h. However, a burst release of about 51% was observed at pH 7.0, indicating a release triggered by diffusion and release of drugs near the surface of the nanoparticles. The slow linear release noted after that could have been caused by the diffusion of 2M4VP from the bulk of the nanoparticle 38 . Controlled drug releases were observed in all media of various pH included in this study, indicating the possibility of achieving a targeted release of drug 2M4VP in a controlled manner. When fitting into different mathematical kinetics models, the cumulative percentage of drug release in-vitro revealed that the release of 2M4VP from the CAP nanoparticles was concentration gradient-based diffusion and followed Fickian law as the n values (release coefficient) was < 0.45 in all the pHs.
The small intestine of humans has a large surface area of ~ 400 m 2 . Once subduing the acidic pH in the stomach, the drug reaching the small intestine faces many physiological barriers, including enzymes, bile, and mucus. The mucus especially could capture and eliminate foreign bodies, which may be beneficial in protecting against gastrointestinal pathogens but could be disadvantageous by limiting drug availability in the small intestine 39,40 . Hence, an orally administrated drug must demonstrate efficient permeability across the mucus and reach the epithelium-a site where V. cholerae adheres and secretes virulence factors for infection 41,42 . Here, the CAP-2M4VP demonstrates a tenfold increased permeability across the intestinal membrane than the pure drug 2M4VP indicating that the polymeric nano-formulation of 2M4VP with CAP could have increased mucus permeability 43 .
Few studies have reported the anti-inflammatory activity of 2M4VP 44,45 . In these studies, various concentrations of 2M4VP demonstrated different levels of cytotoxicity against different cell lines. 2M4VP was reported to have cytotoxicity against Panc-1, while non-cytotoxic to SNU-213 and 293 T cell lines 45 . At concentrations lesser than 40 μM, 2M4VP was non-cytotoxic to RAW264.7 cells. Under LPS-induced conditions, 2M4VP inhibits nitric oxide, prostaglandins, inducible NO synthase, and cyclooxygenase-2 44 . In this study, the CAP-2M4VP at concentrations between 50 and 6.25 μg/mL was non-cytotoxic to Int 407 cell lines indicating the non-toxicity of the nano-formulation at lower concentrations.
Anti-virulence agents supposedly disarm key virulence determinants of a pathogen affecting its infectivity rather than viability (growth). Such agents have an insignificant effect on bacterial cell fitness and do not force bacteria to develop resistance 46,47 . Even at a higher concentration (500 μg/mL), CAP-2M4VP did not express growth inhibitory properties against V. cholerae reference strain (MTCC 3905) and multidrug resistant (MDR) clinical strain (HYR14) at pH 6, 7, 8, and 9. This indicates the non-bactericidal property of the CAP-2M4VP nano-formulation and that the drug targets the QS system and not the growth of the planktonic cell.
As V. cholerae cells are sensitive to gastric acids in the stomach, the biofilm phenotype of V. cholerae protects the cells from acid shock. This enables the cells to evade the acidic pH in the stomach and, upon reaching the small intestine, enhances their colonization at their site of infection. Indeed, the acid tolerance response (ATR) has been identified as the contributing factor for increased colonization of V. cholerae in-vivo 48,49 . Mouse model studies showed that V. cholerae biofilm architecture facilitates virulence. Intact and dispersed V. cholerae biofilm supports more significant colonization outcompeting planktonic cell colonization in-vivo. And the infectious dosage of biofilm cells was also lesser than the planktonic cells 50 . In this study, at 31.25 μg/mL, about 50% inhibition of biofilm formation was noted in both reference and MDR clinical strains at pH 6, 7, 8, and 9. Moreover, fluorescent microscopy images revealed that the treatment of CAP-2M4VP led to the absence of well-established biofilms at all pHs. The biofilm formed by V. cholerae cells in the presence of CAP-2M4VP is thinner and disintegrated at pH 6, 7, 8, and 9. As the biofilm structure is crucial for survival against acidic shock and colonisation 51 , failure to form a mature well-structured biofilm by V. cholerae could affect its survival, intestinal colonization, and hypervirulence property.
Adherence is one of the critical variables affecting V. cholerae colonization in the small intestine. V. cholerae must penetrate the thick mucus barrier in the small intestine using mucinases 52,53 , followed by reversibly and irreversibly attachment to the epithelial cells to first identify a suitable site for infection 54,55 . Various adhesion molecules, including GbpA (GlcNAc-binding protein), facilitates V. cholerae adhesion, and a decrease in the adherence of V. cholerae to epithelium has directly affected the fitness of colonisation 28 . In this study, CAP-2M4VP reduces the adherence of V. cholerae to Int 407 cell lines in-vitro suggesting that CAP-2M4VP could also affect the colonization of V. cholerae, an indispensable stage of V. cholerae life cycle.
Parallel QS circuits function co-ordinately to regulate the expression of virulence in V. cholerae, including the production of biofilm, protease, and cholera toxin through response regulators LuxO and HapR 56 . At low cell density, the phosphorylated LuxO in its active state as a kinase represses hapR to positively regulate the genes encoding biofilm formation and cholera toxin production 7 . Gene expression analysis in the presence of CAP-2M4VP at pH 6, 7, 8, and 9 showed reductions in the expression of genes encoding virulence in different pHs. Overall, in all the pHs the expression of tcp is highly reduced, followed by sRNA qrr-2 and qrr-4, ct, and aphA. The toxin-coregulated pili (TCP) is a type IV pili and vital for the attachment of V. cholerae to enterocytes and M cells 57,58 . TCP also increases the production of MUC4 mucin, which facilitates V. cholerae adhesion right from the initial stages of infections 59 . Here, the notable decrease in the expression of tcp across the pHs indicates that CAP-2M4VP could potentially affect the adherence of V. cholerae in the small intestine. Similarly, the reduction in the expression of ct, a gene encoding the production of cholera toxin, is also observed across all pHs. The QS signal molecules CAI-1 and AI-2 converge at LuxO, which in association with σ N positively regulates the expression of sRNA (qrr 1-4). The sRNAs activate the transcription of aphA, which transcribes the virulence genes tcp and ct. The reduction in the expression of genes encoding qrr2, qrr4, aphA, tcp, and ct indicates downregulation in the expression of all these genes could be possible through the inactivation of LuxO. www.nature.com/scientificreports/ To conclude, the polymeric nano-formulation of 2M4VP encapsulated in cellulose acetate phthalate was evaluated for its potential to inhibit the virulence of V. cholerae. The characterization of polymeric nano-formulation of 2M4VP indicated mono-dispersion and good size distribution. FTIR, XRD, and TGA-DSC analysis showed that chemical interaction between the polymer and the drug was absent, solid-state conversion from the crystalline shape of the drug to amorphous and increase in thermal stability. This indicated polymeric nano-formulation has introduced many biocompatible properties to 2M4VP. In addition to this, the controlled drug release pattern and increase in permeation across the intestinal membrane ex-vivo further emphasize the bio-effectivity of the formulation of 2M4VP in CAP. Furthermore, the formulation is non-bactericidal, non-cytotoxic, inhibits biofilm formation, and adherence, and downregulates the major virulence encoding genes in V. cholerae.

Data availability
The datasets generated during and/or analysed during the current study are available from the corresponding author on reasonable request.